Development of smart cell-free and cell-based assay systems for investigation of leukotriene C4 synthase activity and evaluation of inhibitors

Cysteinyl leukotrienes (cys-LTs) cause bronchoconstriction in anaphylaxis and asthma. They are formed by 5-lipoxygenase (5-LOX) from arachidonic acid (AA) yielding the unstable leukotriene A4 (LTA4) that is subsequently conjugated with glutathione (GSH) by LTC4 synthase (LTC4S). Cys-LT receptor antagonists and LTC4S inhibitors have been developed, but only the former have reached the market. High structural homology to related enzymes and lack of convenient test systems due to instability of added LTA4 have hampered the development of LTC4S inhibitors. We present smart cell-free and cell-based assay systems based on in situ-generated LTA4 that allow studying LTC4S activity and investigating LTC4S inhibitors. Co-incubations of microsomes from HEK293 cells expressing LTC4S with isolated 5-LOX efficiently converted exogenous AA to LTC4 (~ 1.3 μg/200 μg protein). Stimulation of HEK293 cells co-expressing 5-LOX and LTC4S with Ca2 +-ionophore A23187 and 20 μM AA resulted in strong LTC4 formation (~ 250 ng/106 cells). MK-886, a well-known 5-LOX activating protein (FLAP) inhibitor that also acts on LTC4S, consistently inhibited LTC4 formation in all assay types (IC50 = 3.1-3.5 μM) and we successfully confirmed TK04a as potent LTC4S inhibitor in these assay systems (IC50 = 17 and 300 nM, respectively). We demonstrated transcellular LTC4 biosynthesis between neutrophils or 5-LOX-expressing HEK293 cells that produce LTA4 from AA and HEK293 cells expressing LTC4S that transform LTA4 to LTC4. In conclusion, our assay approaches are advantageous as the substrate LTA4 is generated in situ and are suitable for studying enzymatic functionality of LTC4S including site-directed mutations and evaluation of LTC4S inhibitors.